A variety of studies conducted at the ultrastructural and biochemical levels have attempted to elucidate the basic mechanisms of cellular aging by comparing the properties of human diploid cells at early passage in culture to those of late passage cultures. It has become evident, however, that two physiologically distinct subpopulations emerge in senescent cultures of human fibroblasts. The postmitotic cells, comprising one subpopulation, have larger nuclei and a larger modal cell volume than do the actively proliferating fibroblasts which comprise the other subpopulation. Data obtained on whole senescent cultures are therefore representative of a mixed population and do not characterize individually either the actively proliferating or the postmitotic fibroblasts. The objective of this proposed research is to characterize these two subpopulations by measuring the quantitative changes that accompany senescence in human diploid fibroblasts in 1) the volume density (cubic micrometers/cell) of nuclei, nucleoli, mitochondria, smooth and rough endoplasmic reticulum, golgi, lysosomes, secretory granules, pigment granules and lipid droplets, 2) the surface density (square micrometers/cell) of nuclear envelope, smooth and rough endoplasmic reticulum and golgi, 3) the line density (micrometers/cell) of microtubules and microfilaments and 4) the numerical density no./cell) of ribosomes. This will be accomplished in experiments where stereological analyses will be conducted on autoradiograms of early, middle and late passage cultures previously exposed to 3H-TdR. Additionally, the kinetics of transition from the cycling to non-cycling state of fibroblasts aged in vitro will be investigated in an attempt to understand better the growth characteristics of these cells.